Concise Review: Progress and Challenges in Using Human Stem Cells for Biological and Therapeutics Discovery: Neuropsychiatric Disorders.

In facing the daunting challenge of using human embryonic and induced pluripotent stem cells to study complex neural circuit disorders such as schizophrenia, mood and anxiety disorders, and autism spectrum disorders, a 2012 National Institute of Mental Health workshop produced a set of recommendations to advance basic research and engage industry in cell-based studies of neuropsychiatric disorders. This review describes progress in meeting these recommendations, including the development of novel tools, strides in recapitulating relevant cell and tissue types, insights into the genetic basis of these disorders that permit integration of risk-associated gene regulatory networks with cell/circuit phenotypes, and promising findings of patient-control differences using cell-based assays. However, numerous challenges are still being addressed, requiring further technological development, approaches to resolve disease heterogeneity, and collaborative structures for investigators of different disciplines. Additionally, since data obtained so far is on small sample sizes, replication in larger sample sets is needed. A number of individual success stories point to a path forward in developing assays to translate discovery science to therapeutics development.

Viral mitochondria-localized inhibitor of apoptosis (UL37 exon 1 protein) does not protect human neural precursor cells from human cytomegalovirus-induced cell death.

Congenital human cytomegalovirus (HCMV) infection can cause severe brain abnormalities. Apoptotic HCMV-infected brain cells have been detected in a congenitally infected infant. In biologically relevant human neural precursor cells (hNPCs), cultured in physiological oxygen tensions, HCMV infection (m.o.i. of 1 or 3) induced cell death within 3 days post-infection (p.i.) and increased thereafter. Surprisingly, its known anti-apoptotic genes, including the potent UL37 exon 1 protein (pUL37x1) or viral mitochondria-localized inhibitor of apoptosis (vMIA), which protects infected human fibroblasts (HFFs) from apoptosis and from caspase-independent, mitochondrial serine protease-mediated cell death, were expressed by 2 days p.i. Consistent with this finding, an HCMV UL37x1 mutant, BADsubstitutionUL37x1 (BADsubUL37x1) induced cell death in hNPCs (m.o.i. = 1) to level which were indistinguishable from parental virus (BADwild-type)-infected hNPCs. Surprisingly, although BADsubUL37x1 is growth defective in permissive HFFs, it produced infectious progeny in hNPCs with similar kinetics and to levels comparable to BADwild-type-infected hNPCs (m.o.i. = 1). While delayed at a lower multiplicity (m.o.i. = 0.3), the BADsubUL37x1 mutant reached similar levels to revertant within 12 days, in contrast to its phenotype in HFFs. The inability of pUL37x1/vMIA to protect hNPCs from HCMV-induced cell death did not result from impaired trafficking as pUL37x1/vMIA trafficked efficiently to mitochondria in transfected hNPCs and in HCMV-infected hNPCs. These results establish that pUL37x1/vMIA, although protective in permissive HFFs, does not protect HCMV-infected hNPCs from cell death under physiologically relevant oxygen tensions. They further suggest that pUL37x1/vMIA is not essential for HCMV growth in hNPCs and has different cell type-specific roles.

Interaction of hypoxia-inducible factor-1α and Notch signaling regulates medulloblastoma precursor proliferation and fate.

Medulloblastoma (MDB) is the most common brain malignancy of childhood. It is currently thought that MDB arises from aberrantly functioning stem cells in the cerebellum that fail to maintain proper control of self-renewal. Additionally, it has been reported that MDB cells display higher endogenous Notch signaling activation, known to promote the survival and proliferation of neoplastic neural stem cells and to inhibit their differentiation. Although interaction between hypoxia-inducible factor-1α (HIF-1α) and Notch signaling is required to maintain normal neural precursors in an undifferentiated state, an interaction has not been identified in MDB. Here, we investigate whether hypoxia, through HIF-1α stabilization, modulates Notch1 signaling in primary MDB-derived cells. Our results indicate that MDB-derived precursor cells require hypoxic conditions for in vitro expansion, whereas acute exposure to 20% oxygen induces tumor cell differentiation and death through inhibition of Notch signaling. Importantly, stimulating Notch1 activation with its ligand Dll4 under hypoxic conditions leads to expansion of MDB-derived CD133(+) and nestin(+) precursors, suggesting a regulatory effect on stem cells. In contrast, MDB cells undergo neuronal differentiation when treated with γ-secretase inhibitor, which prevents Notch activation. These results suggest that hypoxia, by maintaining Notch1 in its active form, preserves MDB stem cell viability and expansion.

Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.

Hypoxia commonly occurs in solid tumors of the central nervous system (CNS) and often interferes with therapies designed to stop their growth. We found that pediatric high-grade glioma (HGG)-derived precursors showed greater expansion under lower oxygen tension, typical of solid tumors, than normal CNS precursors. Hypoxia inhibited p53 activation and subsequent astroglial differentiation of HGG precursors. Surprisingly, although HGG precursors generated endogenous bone morphogenetic protein (BMP) signaling that promoted mitotic arrest under high oxygen tension, this signaling was actively repressed by hypoxia. An acute increase in oxygen tension led to Smad activation within 30 minutes, even in the absence of exogenous BMP treatment. Treatment with BMPs further promoted astroglial differentiation or death of HGG precursors under high oxygen tension, but this effect was inhibited under hypoxic conditions. Silencing of hypoxia-inducible factor 1alpha (HIF1alpha) led to Smad activation even under hypoxic conditions, indicating that HIF1alpha is required for BMP repression. Conversely, BMP activation at high oxygen tension led to reciprocal degradation of HIF1alpha; this BMP-induced degradation was inhibited in low oxygen. These results show a novel, mutually antagonistic interaction of hypoxia-response and neural differentiation signals in HGG proliferation, and suggest differences between normal and HGG precursors that may be exploited for pediatric brain cancer therapy.

The role of oxygen in regulating neural stem cells in development and disease.

Oxygen (O2) is a substrate for energy production in the cell and is a rapid regulator of cellular metabolism. Recent studies have also implicated O2 and its signal transduction pathways in controlling cell proliferation, fate, and morphogenesis during the development of many tissues, including the nervous system. O2 tensions in the intact brain are much lower than in room air, and there is evidence that dynamic control of O2 availability may be a component of the in vivo neural stem cell (NSC) niche. At lower O2 tensions, hypoxia-inducible factor 1alpha (HIF1alpha) facilitates signal transduction pathways that promote self-renewal (e.g., Notch) and inhibits pathways that promote NSC differentiation or apoptosis (e.g., bone morphogenetic proteins). Increasing O2 tension degrades HIF1alpha, thus promoting differentiation or apoptosis of NSCs and progenitors. These dynamic changes in O2 tension can be mimicked to optimize ex vivo production methods for cell replacement therapies. Conversely, disrupted O2 availability may play a critical role in disease states such as stroke or brain tumor progression. Hypoxia during stroke activates precursor proliferation in vivo, while glioblastoma stem cells proliferate maximally in a more hypoxic environment than normal stem cells, which may make them resistant to certain anti-neoplastic therapies. These findings suggest that O2 response is central to the normal architecture and dynamics of NSC regulation and in the etiology and treatment of brain diseases.

BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells.

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells.

In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His-->Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.

The control of neural stem cells by morphogenic signals.

A complex orchestration of stem-cell specification, expansion and differentiation is required for the proper development of the nervous system. Although progress has been made on the role of individual genes in each of these processes, there are still unresolved questions about how gene function translates to the dynamic assembly of cells into tissues. Recently, stem-cell biology has emerged as a bridge between the traditional fields of cell biology and developmental genetics. In addition to their potential therapeutic role, stem cells are being exploited as experimental 'logic chips' that integrate information and exhibit self-organizing properties. Recent studies provide new insights on how morphogenic signals coordinate major stem cell decisions to regulate the size, shape and cellular diversity of the nervous system.

Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels.

Despite evidence that oxygen regulates neural precursor fate, the effects of changing oxygen tensions on distinct stages in precursor differentiation are poorly understood. We found that 5% oxygen permitted clonal and long-term expansion of mouse fetal cortical precursors. In contrast, 20% oxygen caused a rapid decrease in hypoxia-inducible factor 1alpha and nucleophosmin, followed by the induction of p53 and apoptosis of cells. This led to a decrease in overall cell number and particularly a loss of astrocytes and oligodendrocytes. Clonal analysis revealed that apoptosis in 20% oxygen was due to a complete loss of CD133(lo)CD24(lo) multipotent precursors, a substantial loss of CD133(hi)CD24(lo) multipotent precursors, and a failure of remaining CD133(hi)CD24(lo) cells to generate glia. In contrast, committed neuronal progenitors were not significantly affected. Switching clones from 5% to 20% oxygen only after mitogen withdrawal led to a decrease in total clone numbers but an even greater decrease in oligodendrocyte-containing clones. During this late exposure to 20% oxygen, bipotent glial (A2B5+) and early (platelet-derived growth factor receptor alpha) oligodendrocyte progenitors appeared and disappeared more quickly, relative to 5% oxygen, and late stage O4+ oligodendrocyte progenitors never appeared. These results indicate that multipotent cells and oligodendrocyte progenitors are more susceptible to apoptosis at 20% oxygen than committed neuronal progenitors. This has important implications for optimizing ex vivo production methods for cell replacement therapies.

Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders.

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.

Meeting report: using stem cells for biological and therapeutics discovery in mental illness, April 2012.

This report synthesizes the discussions during a workshop convened April 24-25, 2012, by the National Institute of Mental Health and the Foundation for the NIH in Bethesda, Maryland, that focused on progress and challenges in the use of patient-derived reprogrammed cells for basic biological discovery, target identification, screening, and drug development for mental illnesses such as schizophrenia, bipolar disorder, and autism spectrum disorders. The workshop revealed that the greatest progress has been made in reprogramming methods and agreed-upon standards for validating the resulting induced pluripotent stem cell lines. However, challenges remain in several areas, including efficiently generating and validating specific neural cell types with respect to regional identity, establishing assays with predictive validity to mental illness pathophysiology, and generating sufficient statistical power and data reproducibility across laboratories. A brainstorming session yielded a number of suggestions, including calls to (a) facilitate the replication of results by standardizing protocols and samples used across laboratories; (b) improve technology by generating cheaper/faster targeting methods, reporters, and assays; and (c) improve resource sharing and collaboration, with an emphasis on rapid sharing of new cell lines, technologies, and best practices, possibly incorporated into a public-private partnership. The meeting provided an important venue for academic, government, and private sector scientists to address potential opportunities for translational and clinical applications of reprogrammed cell research. A number of activities since the workshop have reflected the feedback from meeting participants.

Institutional profile: National Institute of Neurological Disorders and Stroke and National Institute of Mental Health.

The nervous system is consistently viewed as a target of high interest for stem cell-based therapeutics. In the USA, the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institute of Mental Health (NIMH) are the two largest funders of neuroscience-related research within the NIH. Research spanning the spectrum of basic, translational and clinical science is conducted both on the NIH campus and through funding of extramural research organizations across the USA, and, to a lesser extent, worldwide. In this brief survey, we will present an overview of stem cell-related activities at the two neuroscience-focused NIH institutes.

Hypoxia and succinate antagonize 2-deoxyglucose effects on glioblastoma.

Glioblastoma multiforme (GBM) are highly proliferative brain tumors characterized by a hypoxic microenvironment which controls GBM stem cell maintenance. Tumor hypoxia promotes also elevated glycolytic rate; thus, limiting glucose metabolism is a potential approach to inhibit tumor growth. Here we investigate the effects mediated by 2-deoxyglucose (2-DG), a glucose analogue, on primary GBM-derived cells maintained under hypoxia. Our results indicate that hypoxia protects GBM cells from the apoptotic effect elicited by 2-DG, which raises succinate dehydrogenase activity thus promoting succinate level decrease. As a consequence hypoxia inducible factor-1α (HIF-1α) degradation occurs and this induces GBM cells to acquire a neuronal committed phenotype. By adding succinate these effects are reverted, as succinate stabilizes HIF-1α and increases GBM stem cell fraction particularly under hypoxia, thus preserving the tumor stem cell niche. 2-DG inhibits anaerobic glycolysis altering GBM cell phenotype by forcing tumor cells into mitochondrial metabolism and by inducing differentiation.

Molecular mechanisms of HIF-1alpha modulation induced by oxygen tension and BMP2 in glioblastoma derived cells.

BACKGROUND: Glioblastoma multiforme (GBM) is one of most common and still poorly treated primary brain tumors. In search for new therapeutic approaches, Bone Morphogenetic Proteins (BMPs) induce astroglial commitment in GBM-derived cells in vitro. However, we recently suggested that hypoxia, which is characteristic of the brain niche where GBM reside, strongly counter-acts BMP effects. It seems apparent that a more complete understanding of the biology of GBM cells is needed, in particular considering the role played by hypoxia as a signaling pathways regulator. HIF-1alpha is controlled at the transcriptional and translational level by mTOR and, alike BMP, also mTOR pathway modulates glial differentiation in central nervous system (CNS) stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate the role of mTOR signaling in the regulation of HIF-1alpha stability in primary GBM-derived cells maintained under hypoxia (2% oxygen). We found that GBM cells, when acutely exposed to high oxygen tension, undergo Akt/mTOR pathway activation and that BMP2 acts in an analogous way. Importantly, repression of Akt/mTOR signaling is maintained by HIF-1alpha through REDD1 upregulation. On the other hand, BMP2 counter-acts HIF-1alpha stability by modulating intracellular succinate and by controlling proline hydroxylase 2 (PHD2) protein through inhibition of FKBP38, a PHD2 protein regulator. CONCLUSIONS/SIGNIFICANCE: In this study we elucidate the molecular mechanisms by which two pro-differentiating stimuli, BMP2 and acute high oxygen exposure, control HIF-1alpha stability. We previously reported that both these stimuli, by inducing astroglial differentiation, affect GBM cells growth. We also found differences in high oxygen and BMP2 sensitivity between GBM cells and normal cells that should be further investigated to better define tumor cell biology.

Potency and fate specification in CNS stem cell populations in vitro.

To realize the promise of stem cell biology, it is important to identify the precise time in the history of the cell when developmental potential is restricted. To achieve this goal, we developed a real-time imaging system that captures the transitions in fate, generating neurons, astrocytes, and oligodendrocytes from single CNS stem cells in vitro. In the presence of bFGF, tripotent cells normally produce specified progenitors through a bipotent intermediate cell type. Surprisingly, the tripotent state is reset at each passage. The cytokine CNTF is thought to instruct multipotent cells to an astrocytic fate. We demonstrate that CNTF both directs astrogliogenesis from tripotent cells, bypassing two of the three normal bipotent intermediates, and later promotes the expansion of specified astrocytic progenitors. These results show how discrete cell types emerge from a multipotent cell and provide a strong basis for future studies to determine the molecular basis of fate specification.

Concise review: bone morphogenetic protein pleiotropism in neural stem cells and their derivatives--alternative pathways, convergent signals.

Bone morphogenetic proteins (BMPs) are a class of morphogens that are critical regulators of the central nervous system (CNS), peripheral nervous system, and craniofacial development. Modulation of BMP signaling also appears to be an important component of the postnatal stem cell niche. However, describing a comprehensive model of BMP actions is complicated by their paradoxical effects in precursor cells, which include dorsal specification, promoting proliferation or mitotic arrest, cell survival or death, and neuronal or glial fate. In addition, in postmitotic neurons BMPs can promote dendritic growth, act as axonal chemorepellants, and stabilize synapses. Although many of these responses depend on interactions with other incoming signals, some reflect the recruitment of distinct BMP signal transduction pathways. In this review, we classify the diverse effects of BMPs on neural cells, focus on the known mechanisms that specify distinct responses, and discuss the remaining challenges in identifying the cellular basis of BMP pleiotropism. Addressing these issues may have importance for stem cell mobilization, differentiation, and cell integration/survival in reparative therapies.

Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24.

Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24(hi) population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment. Disclosure of potential conflicts of interest is found at the end of this article.

Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.

Human neural precursor proliferation and potency is limited by senescence and loss of oligodendrocyte potential. We found that in vitro expansion of human postnatal brain CD133(+) nestin(+) precursors is enhanced at 5% oxygen, while raising oxygen tension to 20% depletes precursors and promotes astrocyte differentiation even in the presence of mitogens. Higher cell densities yielded more astrocytes regardless of oxygen tension. This was reversed by noggin at 5%, but not 20%, oxygen due to a novel repressive effect of low oxygen on bone morphogenetic protein (BMP) signaling. When induced to differentiate by mitogen withdrawal, 5% oxygen-expanded precursors generated 17-fold more oligodendrocytes than cells expanded in 20% oxygen. When precursors were expanded at 5% oxygen and then differentiated at 20% oxygen, oligodendrocyte maturation was further enhanced 2.5-fold. These results indicate that dynamic control of oxygen tension regulates different steps in fate and maturation and may be crucial for treating neurodegenerative diseases.

BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells.

CNS stem cells are best characterized by their ability to self-renew and to generate multiple differentiated derivatives, but the effect of mitogenic signals, such as fibroblast growth factor 2 (FGF2), on the positional identity of these cells is not well understood. Here, we report that bone morphogenetic protein 2 (BMP2) induces telencephalic CNS stem cells to fates characteristic of neural crest and choroid plexus mesenchyme, a cell type of undetermined lineage in rodents. This induction occurs both in dissociated cell culture and cortical explants of embryonic day 14.5 (E14.5) embryos, but only when cells have been exposed to FGF2. Neither EGF nor IGF1 can substitute for FGF2. An early step in this response is activation of beta-catenin, a mediator of Wnt activity. The CNS stem cells first undergo an epithelial-to-mesenchymal transition and subsequently differentiate to smooth-muscle and non-CNS glia cells. Similar responses are seen with stem cells from E14.5 cortex, E18.5 cortex and adult subventricular zone, but with a progressive shift toward gliogenesis that is characteristic of normal development. These data indicate that FGF2 confers competence for dorsalization independently of its mitogenic action. This rapid and efficient induction of dorsal fates may allow identification of positional identity effectors that are co-regulated by FGF2 and BMP2.